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Summary Kline et al. (1980) have reported that indole-3-acetic acid (IAA) and four other indole derivatives are able to substitute for cAMP in activating expression of the ara regulon of E. coli. We have examined this phenomenon in detail, utilizing fusions between the structural gene for β-galactosidase and the promoters for the araBAD, araE, and araFG operons. We confirm that IAA potently stimulates transcription from the araBAD promoter. The effect is highly specific to araBAD, as IAA has no, or only slight, effects on the araE and araFG operons. However, contrary to the results of Kline et al., we find that the action of IAA does not require CAP. Thus, IAA fully stimulates the transcription of araBAD in a strain which bears a complete deletion of the crp gene.

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[Auszug] Three mutations that alter the DNA sequence specificity of the catabolite gene activator protein (CAP) from AA-TGTGA--T---TCA-ATW to AA-TGTAA--T---TCA-ATW have been isolated. All three mutations affect the same amino acid of CAP, glutamic acid 181. We propose that it is this amino acid of CAP that ...

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[Auszug] The σ70 subunit of RNA polymerase plays an essential role in transcription initiation. In addition, σ70 has a critical regulatory role during transcription elongation at the bacteriophage λ late promoter, λPR′. At this promoter, σ70 mediates a pause in early ...

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[Auszug] 5-Bromouracil (BrU) is an isosteric photoreactive analogue of thymine (Fig. I)13. We have incorporated BrU at a single position within the DNA site for GCN4, prepared the protein-DNA complex, irradiated the protein-DNA complex with ultra-violet light and determined the amino acid at which ...

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[Auszug] We have used the following components: (1) CAP, (2) a fluorochrome-labelled 42-base-pair oligodeoxyribonucleotide duplex containing the consensus DNA site for CAP24 and containing no known specific determinants for binding of RNA polymerase (TCAP42FL'; Fig. 1), and (3) Escherichia coli RNA ...

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[Auszug] Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively ...

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Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how a single activator molecule can make multiple interactions with the transcription machinery, with each interaction being responsible for a specific mechanistic consequence. At Class II CAP-dependent promoters, the DNA target site for CAP is centred near position −42, overlapping and replacing the −35 determinant for binding of RNA polymerase (RNAP). Transcription activation requires two distinct mechanistic components. The first component is ‘anti-inhibition,’ overcoming an inhibitory effect of the RNAP α subunit C-terminal domain (αCTD). This component involves direct contact between amino acids 156–164 (activating region 1) of the upstream subunit of the CAP dimer and a target in αCTD. The second component is ‘direct activation’, facilitating isomerization of the RNAP–promoter closed complex to the transcriptionally competent open complex. This component involves direct contact between amino acids 19, 21 and 101 (activating region 2) of the downstream subunit of the CAP dimer and a target in the RNAP α subunit N-terminal domain (αNTD).

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[Auszug] Sir—Escherichia coli catabolite activator protein (CAP) binds to DNA as a dimer of two identical protomers and makes two-fold symmetric interactions with a 22 bp two-fold symmetric DNA site (reviewed in Ref. 1). In the CAP–DNA complex, the carboxylate side chain of Glu 181 of the CAP ...